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1.  Use of type I interferon-inducible mRNAs as pharmacodynamic markers and potential diagnostic markers in trials with sifalimumab, an anti-IFNα antibody, in systemic lupus erythematosus 
Arthritis Research & Therapy  2010;12(Suppl 1):S6.
Type I interferons are implicated in the pathogenesis of systemic lupus erythematosus (SLE). Type I interferon-inducible mRNAs are widely and concordantly overexpressed in the periphery and involved tissues of a subset of SLE patients, and provide utility as pharmacodynamic biomarkers to aid dose selection, as well as potential indicators of patients who might respond favorably to anti-IFNα therapy in SLE. We implemented a three-tiered approach to identify a panel of type I interferon-inducible mRNAs to be used as potential pharmacodynamic biomarkers to aid dose selection in clinical trials of sifalimumab, an anti-IFNα monoclonal antibody under development for the treatment of SLE. In a single-dose escalation phase 1 trial, we observed a sifalimumab-specific and dose-dependent inhibition of the overexpression of type I interferon-inducible mRNAs in the blood of treated subjects. Inhibition of expression of type I interferon-inducible mRNAs and proteins was also observed in skin lesions of SLE subjects from the same trial. Inhibiting IFNα resulted in a profound downstream effect in these SLE subjects that included suppression of mRNAs of B-cell activating factor belonging to the TNF family and the signaling pathways of TNFα, IL-10, IL-1β, and granulocyte-macrophage colony-stimulating factor in both the periphery and skin lesions. A scoring method based on the expression of type I interferon-inducible mRNAs partitioned SLE patients into two distinct subpopulations, which suggests the possibility of using these type I interferon-inducible genes as predictive biomarkers to identify SLE patients who might respond more favorably to anti-type I interferon therapy.
doi:10.1186/ar2887
PMCID: PMC2991779  PMID: 20392292
2.  Genetic Variation near IRF8 is Associated with Serologic and Cytokine Profiles in Systemic Lupus Erythematosus and Multiple Sclerosis 
Genes and immunity  2013;14(8):10.1038/gene.2013.42.
Alleles of IRF8 are associated with susceptibility to both systemic lupus erythematosus (SLE) and multiple sclerosis (MS). While high type I interferon (IFN) is thought to be causal in SLE, type I IFN is used as a therapy in MS. We investigated whether IRF8 alleles were associated with type I IFN levels or serologic profiles in SLE and MS. Alleles which have been previously associated with SLE or MS were genotyped in SLE and MS patients. The MS-associated rs17445836G allele was associated with anti-dsDNA autoantibodies in SLE patients (meta-analysis OR=1.92). The same allele was associated with decreased serum IFN activity in SLE patients with anti-dsDNA antibodies, and with decreased type I IFN-induced gene expression in PBMC from anti-dsDNA negative SLE patients. In secondary progressive MS patients, rs17445836G was associated with decreased serum type I IFN. Rs17445836G was associated with increased IRF8 expression in SLE patient B cells. In summary, IRF8 rs17445836G is associated with human autoimmune disease characterized by low type I IFN levels, and this may have pharmacogenetic relevance as type I IFN is modulated in SLE and MS. The association with autoantibodies and increased IRF8 expression in B cells supports a role for rs17445836G in humoral tolerance.
doi:10.1038/gene.2013.42
PMCID: PMC3856198  PMID: 23965942
systemic lupus erythematosus; type I interferon; autoantibodies; interferon regulatory factors
3.  Activation of the Interferon Pathway is Dependent Upon Autoantibodies in African-American SLE Patients, but Not in European-American SLE Patients 
Background: In systemic lupus erythematosus (SLE), antibodies directed at RNA-binding proteins (anti-RBP) are associated with high serum type I interferon (IFN), which plays an important role in SLE pathogenesis. African-Americans (AA) are more likely to develop SLE, and SLE is also more severe in this population. We hypothesized that peripheral blood gene expression patterns would differ between AA and European-American (EA) SLE patients, and between those with anti-RBP antibodies and those who lack these antibodies.
Methods: Whole blood RNA from 33 female SLE patients and 16 matched female controls from AA and EA ancestral backgrounds was analyzed on Affymetrix Gene 1.0 ST gene expression arrays. Ingenuity Pathway Analysis was used to compare the top differentially expressed canonical pathways amongst the sample groups. An independent cohort of 116 SLE patients was used to replicate findings using quantitative real-time PCR (qPCR).
Results: Both AA and EA patients with positive anti-RBP antibodies showed over-expression of similar IFN-related canonical pathways, such as IFN Signaling (P = 1.3 × 10−7 and 6.3 × 10−11 in AA vs. EA respectively), Antigen Presenting Pathway (P = 1.8 × 10−5 and 2.5 × 10−6), and a number of pattern recognition receptor pathways. In anti-RBP negative (RBP−) patients, EA subjects demonstrated similar IFN-related pathway activation, whereas no IFN-related pathways were detected in RBP−AA patients. qPCR validation confirmed similar results.
Conclusion: Our data show that IFN-induced gene expression is completely dependent on the presence of autoantibodies in AA SLE patients but not in EA patients. This molecular heterogeneity suggests differences in IFN-pathway activation between ancestral backgrounds in SLE. This heterogeneity may be clinically important, as therapeutics targeting this pathway are being developed.
doi:10.3389/fimmu.2013.00309
PMCID: PMC3787392  PMID: 24101921
systemic lupus erythematosus; interferon alpha; autoantibodies; ancestral background; interferon gamma
4.  The innate immune system in SLE: type I interferons and dendritic cells 
Lupus  2008;17(5):394-399.
Patients with systemic lupus erythematosus (SLE) have an increased expression of type I interferon (IFN) regulated genes because of a continuous production of IFN-α. The cellular and molecular background to this IFN-α production has started to be elucidated during the last years, as well as the consequences for the innate and adaptive immune systems. Plasmacytoid dendritic cells (pDC) activated by immune complexes containing nucleic acids secrete type I IFN in SLE. Type I IFN causes differentiation of monocytes to myeloid-derived dendritic cell (mDC) and activation of auto-reactive T and B cells. A new therapeutic option in patients with SLE is, therefore, inhibition of IFN-α, and recent data from a phase I clinical trial suggests that administration of neutralizing monoclonal antibodies against anti-IFN-α can ameliorate disease activity.
doi:10.1177/0961203308090020
PMCID: PMC3694565  PMID: 18490415
5.  Flow cytometry analysis of glucocorticoid receptor expression and binding in steroid-sensitive and steroid-resistant patients with systemic lupus erythematosus 
Arthritis Research & Therapy  2009;11(4):R108.
Introduction
Glucocorticoid (GC) therapy is the main treatment for systemic lupus erythematosus (SLE). However, some patients are resistant to these agents. Abnormalities of glucocorticoid receptor (GR) seem to be related to steroid resistance. This study evaluated GRs in T lymphocytes and monocytes of SLE patients by flow cytometry (FCM) using a monoclonal antibody (mAb) and FITC-Dex probes.
Methods
Thirty-five patients with SLE before treatment and 27 age- and sex-matched normal controls were studied. Disease activity scores were determined before and after treatment and used to divide the patients into steroid-resistant (SR) and steroid-sensitive (SS) groups. GRs in T lymphocytes (CD3+) and monocytes (CD14+) were examined by FCM with GR-mAb and FITC-Dex probes before treatment. Peripheral blood mononuclear cells (PBMCs) were isolated for in vitro GCs sensitivity assays. The validity of FCM analysis of intracellular staining for GR with GR-mAb and FITC-Dex probes was evaluated through comparison with western blot and radioligand binding assay (RLBA) in U937 and K562 cells in vitro. One-way ANOVA, student's t test, linear regression and spearman correlation were performed.
Results
A significant decrease in GR binding and the expression in K562 and U937 cells with 10-6 M dexamethasone (Dex) was found compared with those without Dex. In addition, a positive correlation was found between FCM and RLBA as well as FCM and Western blot. The expression and binding of both CD3/GR and CD14/GR in SR patients with SLE, detected by FCM, were all lower than those in SS patients with SLE, whereas there was no significant difference in SS patients and controls. In vitro corticosteroid sensitivity assay indicated that PHA-stimulated tumour necrosis factor-α (TNF-α), IL-12 and interferon-γ (IFN-γ) secretion was significantly inhibited by 10-6 M Dexamethasone in all controls and SS patients, compared with that in SR group, which confirms patient classification as SR and SS by disease activity index (SLEDAI) score.
Conclusions
Abnormalities of expression and binding of the GR may be involved in tissue resistance to steroids in SLE patients. Determination of GR expression and binding by FCM may be useful in predicting the response to steroid treatment of SLE patients.
Trial registration
Clinical trial registration number NCT00600652.
doi:10.1186/ar2763
PMCID: PMC2745790  PMID: 19594946
6.  Constitutive Phosphorylation of Interferon Receptor A-Associated Signaling Proteins in Systemic Lupus Erythematosus 
PLoS ONE  2012;7(7):e41414.
Background
Overexpression of type I interferon (IFN-I)-induced genes is a common feature of systemic lupus erythematosus (SLE) and its experimental models, but the participation of endogenous overproduction of IFN-I on it is not clear. To explore the possibility that abnormally increased IFN-I receptor (IFNAR) signaling could participate in IFN-I-induced gene overexpression of SLE, we examined the phosphorylation status of the IFNAR-associated signaling partners Jak1 and STAT2, and its relation with expression of its physiologic inhibitor SOCS1 and with plasma levels of IFNα and IFN-like activity.
Methodology/Principal Findings
Peripheral blood mononuclear cells (PBMC) from SLE patients with or without disease activity and healthy controls cultured in the presence or in the absence of IFNβ were examined by immunoprecipitation and/or western blotting for expression of the two IFNAR chains, Jak1, Tyk2, and STAT2 and their phosphorylated forms. In SLE but not in healthy control PBMC, Jak1 and STAT2 were constitutively phosphorylated, even in the absence of disease activity (basal pJak1: controls vs. active SLE p<0.0001 and controls vs. inactive SLE p = 0.0006; basal pSTAT2: controls vs. active and inactive SLE p<0.0001). Although SOCS1 protein was slightly but significantly decreased in SLE in the absence or in the presence of IFNβ (p = 0.0096 to p<0.0001), in SOCS1 mRNA levels were markedly decreased (p = 0.036 to p<0.0001). IFNβ induced higher levels of the IFN-I-dependent MxA protein mRNA in SLE than in healthy controls, whereas the opposite was observed for SOCS1. Although there was no relation to increased serum IFNα, active SLE plasma could induce expression of IFN-dependent genes by normal PBMC.
Conclusions/Significance
These findings suggest that in some SLE patients IFN-I dependent gene expression could be the result of a low IFNAR signaling threshold.
doi:10.1371/journal.pone.0041414
PMCID: PMC3408474  PMID: 22859983
7.  Netting Neutrophils Are Major Inducers of Type I IFN Production in Pediatric Systemic Lupus Erythematosus 
Science translational medicine  2011;3(73):73ra20.
Systemic lupus erythematosus (SLE) is a systemic autoimmune disease characterized by a breakdown of tolerance to nuclear antigens and the development of immune complexes. Genomic approaches have shown that human SLE leukocytes homogeneously express type I interferon (IFN)–induced and neutrophil-related transcripts. Increased production and/or bioavailability of IFN-α and associated alterations in dendritic cell (DC) homeostasis have been linked to lupus pathogenesis. Although neutrophils have long been shown to be associated with lupus, their potential role in disease pathogenesis remains elusive. Here, we show that mature SLE neutrophils are primed in vivo by type I IFN and die upon exposure to SLE-derived anti-ribonucleoprotein antibodies, releasing neutrophil extracellular traps (NETs). SLE NETs contain DNA as well as large amounts of LL37 and HMGB1, neutrophil proteins that facilitate the uptake and recognition of mammalian DNA by plasmacytoid DCs (pDCs). Indeed, SLE NETs activate pDCs to produce high levels of IFN-α in a DNA- and TLR9 (Toll-like receptor 9)–dependent manner. Our results reveal an unsuspected role for neutrophils in SLE pathogenesis and identify a novel link between nucleic acid–recognizing antibodies and type I IFN production in this disease.
doi:10.1126/scitranslmed.3001201
PMCID: PMC3143837  PMID: 21389264
8.  Interferon and Granulopoiesis Signatures in Systemic Lupus Erythematosus Blood 
Systemic lupus erythematosus (SLE) is a prototype systemic autoimmune disease characterized by flares of high morbidity. Using oligonucleotide microarrays, we now show that active SLE can be distinguished by a remarkably homogeneous gene expression pattern with overexpression of granulopoiesis-related and interferon (IFN)-induced genes. Using the most stringent statistical analysis (Bonferroni correction), 15 genes were found highly up-regulated in SLE patients, 14 of which are targets of IFN and one, defensin DEFA-3, a major product of immature granulocytes. A more liberal correction (Benjamini and Hochberg correction) yielded 18 additional genes, 12 of which are IFN-regulated and 4 granulocyte-specific. Indeed immature neutrophils were identified in a large fraction of SLE patients white blood cells. High dose glucocorticoids, a standard treatment of disease flares, shuts down the interferon signature, further supporting the role of this cytokine in SLE. The expression of 10 genes correlated with disease activity according to the SLEDAI. The most striking correlation (P < 0.001, r = 0.55) was found with the formyl peptide receptor-like 1 protein that mediates chemotactic activities of defensins. Therefore, while the IFN signature confirms the central role of this cytokine in SLE, microarray analysis of blood cells reveals that immature granulocytes may be involved in SLE pathogenesis.
doi:10.1084/jem.20021553
PMCID: PMC2193846  PMID: 12642603
microarray; immature granulocytes; glucocorticoid; leukocytes; autoimmunity
9.  Association of Endogenous Anti–Interferon-α Autoantibodies With Decreased Interferon-Pathway and Disease Activity in Patients With Systemic Lupus Erythematosus 
Arthritis and rheumatism  2011;63(8):2407-2415.
Objective
Numerous observations implicate interferon-α (IFNα) in the pathophysiology of systemic lupus erythematosus (SLE); however, the potential impact of endogenous anti-IFNα autoantibodies (AIAAs) on IFN-pathway and disease activity is unclear. The aim of this study was to characterize IFN-pathway activity and the serologic and clinical profiles of AIAA-positive patients with SLE.
Methods
Sera obtained from patients with SLE (n = 49), patients with rheumatoid arthritis (n = 25), and healthy control subjects (n = 25) were examined for the presence of AIAAs, using a biosensor immunoassay. Serum type I IFN bioactivity and the ability of AIAA-positive sera to neutralize IFNα activity were determined using U937 cells. Levels of IFN-regulated gene expression in peripheral blood were determined by microarray, and serum levels of BAFF, IFN-inducible chemokines, and other autoantibodies were measured using immunoassays.
Results
AIAAs were detected in 27% of the serum samples from patients with SLE, using a biosensor immunoassay. Unsupervised hierarchical clustering analysis identified 2 subgroups of patients, IFNlow and IFNhigh, that differed in the levels of serum type I IFN bioactivity, IFN-regulated gene expression, BAFF, anti-ribosomal P, and anti-chromatin autoantibodies, and in AIAA status. The majority of AIAA-positive patients had significantly lower levels of serum type I IFN bioactivity, reduced downstream IFN-pathway activity, and lower disease activity compared with the IFNhigh patients. AIAA-positive sera were able to effectively neutralize type I IFN activity in vitro.
Conclusion
Patients with SLE commonly harbor AIAAs. AIAA-positive patients have lower levels of serum type I IFN bioactivity and evidence for reduced downstream IFN-pathway and disease activity. AIAAs may influence the clinical course in SLE by blunting the effects produced by IFNα.
doi:10.1002/art.30399
PMCID: PMC4028124  PMID: 21506093
10.  Monoclonal Antibodies for Systemic Lupus Erythematosus (SLE) † 
Pharmaceuticals  2010;3(1):300-322.
A number of monoclonal antibodies (mAb) are now under investigation in clinical trials to assess their potential role in Systemic Lupus Erythematosus (SLE). The most frequently used mAb is rituximab, which is directed against CD20, a membrane protein expressed on B lymphocytes. Uncontrolled trials reported an improvement of SLE activity in non-renal patients and other studies even reported an improvement of severe lupus nephritis unresponsive to conventional treatments. However two randomized trials failed to show the superiority of rituximab over conventional treatment in non renal SLE and in lupus nephritis. Preliminary trials reported promising results with epratuzumab, a humanized mAb directed against CD22, and with belimumab, a human mAb that specifically recognizes and inhibits the biological activity of BLyS a cytokine of the tumor-necrosis-factor (TNF) ligand superfamily. Other clinical trials with mAb directed against TNF-alpha, interleukin-10 (Il-10), Il-6, CD154, CD40 ligand, IL-18 or complement component C5 are under way. At present, however, in spite of good results reported by some studies, no firm conclusion on the risk-benefit profile of these mAbs in patients with SLE can be drawn from the available studies.
doi:10.3390/ph3010300
PMCID: PMC3991031
systemic lupus erythematosus; lupus nephritis; monoclonal antibodies; rituximab
11.  IRF5 activation in monocytes of SLE patients is triggered by circulating autoantigens independent of type I IFN 
Arthritis and Rheumatism  2012;64(3):788-798.
Objective
Genetic variants of interferon regulatory factor 5 (IRF5) are associated with susceptibility to systemic lupus erythematosus (SLE). IRF5 regulates the expression of proinflammatory cytokines and type I interferons (IFN) believed to be involved in SLE pathogenesis. The aim of this study was to determine the activation status of IRF5 by assessing its nuclear localization in immune cells of SLE patients and healthy donors, and to identify SLE triggers of IRF5 activation.
Methods
IRF5 nuclear localization in subpopulations of peripheral blood mononuclear cells (PBMC) from 14 genotyped SLE patients and 11 healthy controls was assessed using imaging flow cytometry. IRF5 activation and function were examined after ex vivo stimulation of healthy donor monocytes with SLE serum or components of SLE serum. Cellular localization was determined by ImageStream and cytokine expression by Q-PCR and ELISA.
Results
IRF5 was activated in a cell type-specific manner; monocytes of SLE patients had constitutively elevated levels of nuclear IRF5 compared to NK and T cells. SLE serum was identified as a trigger for IRF5 nuclear accumulation; however, neither IFNα nor SLE immune complexes could induce nuclear localization. Instead, autoantigens comprised of apoptotic/necrotic material triggered IRF5 nuclear accumulation in monocytes. Production of cytokines IFNα, TNFα and IL6 in monocytes stimulated with SLE serum or autoantigens was distinct yet correlated with the kinetics of IRF5 nuclear localization.
Conclusion
This study provides the first formal proof that IRF5 activation is altered in monocytes of SLE patients that is in part contributed by the SLE blood environment.
doi:10.1002/art.33395
PMCID: PMC3288585  PMID: 21968701
12.  Belimumab in systemic lupus erythematosus: an update for clinicians 
Systemic lupus erythematosus (SLE) is a chronic inflammatory disorder that is driven by autoantibodies that target multiple organ systems. B-lymphocyte stimulator (BLyS) and its receptors on B-cell subsets play an important role in autoimmune B-cell development and SLE pathogenesis. Targeted therapy with belimumab, the monoclonal antibody against BLyS, has shown clinical benefit in two large-scale, multicenter phase III trials leading to US Food and Drug Administration approval for patients with serologically positive SLE who have active disease despite standard therapy. This review will discuss the challenges in lupus drug development and clinical trials, the basics of B-cell pathogenesis in SLE, the recent lupus clinical trials of B-cell targeted treatments, and other potential targeted therapies under investigation for patients with lupus.
doi:10.1177/2040622311424806
PMCID: PMC3513897  PMID: 23251765
belimumab; B-lymphocyte stimulator; systemic lupus erythematosus
13.  Agrin Signalling Contributes to Cell Activation and Is Overexpressed in T Lymphocytes from Lupus Patients1 
It is shown in this study that the heparan sulfate proteoglycan agrin is overexpressed in T cells isolated from patients with the autoimmune disease systemic lupus erythematosus (SLE). Freshly isolated CD4+ and CD8+ subpopulations both exhibited higher expression over healthy controls, which however, gradually declined when cells were cultured in vitro. Agrin expression was induced following in vitro activation of cells via their Ag receptor, or after treatment with IFN-α, a cytokine shown to be pathogenic in lupus. Furthermore, serum from SLE patients with active disease was able to induce agrin expression when added to T cells from healthy donors, an increase that was partially blocked by neutralizing anti-IFN-α Abs. Cross-linking agrin with mAbs resulted in rapid reorganization of the actin cytoskeleton, activation of the ERK MAPK cascade, and augmentation of anti-CD3-induced proliferation and IL-10 production, indicating that agrin is a functional receptor in T cells. These results demonstrate that agrin expression in human T cells is regulated by cell activation and IFN-α, and may have an important function during cell activation with potential implications for autoimmunity.
PMCID: PMC2596879  PMID: 18025246
14.  Interferon-lambda1 induces peripheral blood mononuclear cell-derived chemokines secretion in patients with systemic lupus erythematosus: its correlation with disease activity 
Introduction
Systemic lupus erythematosus (SLE) is an autoimmune disease involving multiple organ systems. Previous studies have suggested that interferon-lambda 1 (IFN-λ1), a type III interferon, plays an immunomodulatory role. In this study we investigated its role in SLE, including its correlation with disease activity, organ disorder and production of chemokines.
Methods
We determined levels of IFN-λ1 mRNA in peripheral blood mononuclear cells (PBMC) and serum protein levels in patients with SLE using real-time polymerase chain reaction (real-time PCR) and enzyme-linked immunoassay (ELISA). Further, we detected the concentration of IFN-inducible protein-10 (IP-10), monokine induced by IFN-γ (MIG) and interleukin-8 (IL-8) secreted by PBMC under the stimulation of IFN-λ1 using ELISA.
Results
IFN-λ1 mRNA and serum protein levels were higher in patients with SLE compared with healthy controls. Patients with active disease showed higher IFN-λ1 mRNA and serum protein levels compared with those with inactive disease as well. Serum IFN-λ1 levels were positively correlated with Systemic Lupus Erythematosus Disease Activity Index (SLEDAI), anti-dsDNA antibody, C-reactive protein (CRP) and negatively correlated with complement 3. Serum IFN-λ1 levels were higher in SLE patients with renal involvement and arthritis compared with patients without the above-mentioned manifestations. IFN-λ1 with different concentrations displayed different effects on the secretion of the chemokines IP-10, MIG and IL-8.
Conclusions
These findings indicate that IFN-λ1 is probably involved in the renal disorder and arthritis progression of SLE and associated with disease activity. Moreover, it probably plays an important role in the pathogenesis of SLE by stimulating secretion of the chemokines IP-10, MIG and IL-8. Thus, IFN-λ1 may provide a novel research target for the pathogenesis and therapy of SLE.
doi:10.1186/ar3363
PMCID: PMC3218903  PMID: 21679442
15.  A Novel Type I Interferon-producing Cell Subset in Murine Lupus 
Excess type-I interferons (IFN-I) have been linked to the pathogenesis of systemic lupus erythematosus (SLE). Therapeutic use of IFN-I can trigger the onset of SLE and most lupus patients display upregulation of a group of interferon stimulated genes (ISGs). While this “interferon signature” has been linked with disease activity, kidney involvement, and autoantibody production, the source of IFN-I production in SLE remains unclear. Tetramethylpentadecane (TMPD)-induced lupus is at present the only model of SLE associated with excess IFN-I production and ISG expression. Here we demonstrate that TMPD treatment induces an accumulation of immature Ly6Chi monocytes, which are a major source of IFN-I in this lupus model. Importantly, they were distinct from interferon-producing dendritic cells. The expression of IFN-I and ISGs was rapidly abolished by monocyte depletion whereas systemic ablation of dendritic cells (DCs) had little effect. In addition, there was a striking correlation between the numbers of Ly6Chi monocytes and the production of lupus autoantibodies. Therefore, immature monocytes rather than DCs appear to be the primary source of IFN-I in this model of IFN-I dependent lupus.
PMCID: PMC2909121  PMID: 18354236
autoimmunity; systemic lupus erythematosus; monocytes
16.  Nonerosive arthritis in lupus is mediated by IFN-α stimulated monocyte differentiation that is nonpermissive of osteoclastogenesis 
Arthritis and rheumatism  2010;62(4):1127-1137.
Objective
In contrast to rheumatoid arthritis (RA), Jaccoud arthritis (JA) joint inflammation in systemic lupus erythematosus (SLE) is nonerosive. Although the mechanism responsible is unknown, the anti-osteoclastogenic cytokine interferon-alpha (IFN-α), whose transcriptome is present in SLE monocytes, may be responsible. To test this, we examined effects of IFN-α versus lupus disease on osteoclasts and erosion in the NZBxNZW F1 SLE mouse model with K/BxN serum-induced arthritis (SIA).
Methods
Elevated systemic IFN-α levels were obtained by administration of an adenoviral vector expressing IFN-α (Ad-IFN-α). SLE disease was marked by anti-dsDNA antibody titer and proteinuria, and Ifi202 and Mx1 expression represented the IFN-α transcriptome. Micro-CT was used to evaluate bone erosions. Flow cytometry for CD11b and CD11c was used to evaluate the frequency of circulating osteoclast precursors (OCP) and myeloid dendritic cells (mDC) in blood.
Results
Administration of Ad-IFN-α to NZBxNZW F1 mice induced osteopetrosis. Pre-autoimmune NZBxNZW F1 mice are fully susceptible to focal erosions in the setting of SIA. However, NZBxNZW F1 mice with high anti-dsDNA antibody titers and the IFN-α transcriptome were protected against bone erosions. Ad-IFN-α pre-treatment of NZW mice before K/BxN serum administration also resulted in protection against bone erosion (r2=0.4720, p<0.01), which was associated with a decrease in circulating CD11b+CD11c− OCP, and a concomitant increase in CD11b+CD11c+ cells (r2=0.6330, p<0.05) that are phenotypic of mDC.
Conclusion
These findings suggest that IFN-α in SLE shifts monocyte development toward mDC at the expense of osteoclastogenesis thereby resulting in decreased bone erosion.
doi:10.1002/art.27312
PMCID: PMC2854832  PMID: 20131244
Jaccoud arthritis (JA); Lupus; Osteoclast; Interferon-alpha (IFN-α)
17.  Novel Evidence-Based Systemic Lupus Erythematosus Responder Index 
Arthritis and rheumatism  2009;61(9):1143-1151.
Objective
To describe a new systemic lupus erythematosus (SLE) Responder Index (SRI) based on the belimumab phase II SLE trial and demonstrate its potential utility in SLE clinical trials.
Methods
Data from a 449-patient randomized, double-blind, placebo-controlled study of 3 doses of belimumab (1, 4, 10 mg/kg) or placebo plus standard of care therapy (SOC) over a 56-week period were analyzed. SELENA-SLEDAI and BILAG SLE disease activity instruments, SF-36 Health Survey, and biomarker analyses were used to create a novel SRI. Response to treatment in a subset of SLE patients (n=321) who were serologically active (ANA ≥1:80 and/or anti-dsDNA antibody ≥30 IU) at baseline was retrospectively evaluated using the SRI.
Results
SRI response is defined as: 1) ≥4-point reduction in SELENA-SLEDAI score; 2) no new BILAG A or no more than 1 new BILAG B domain score; and 3) no deterioration from baseline in the Physician’s Global Assessment (PGA) by ≥0.3 points. In serologically active patients, addition of belimumab to SOC resulted in a response in 46% of patients at week 52 compared with 29% for the placebo patients (P=0.006). SRI responses were independent of baseline autoantibody subtype.
Conclusion
Evidence-based evaluation of a large randomized, placebo-controlled trial in SLE resulted in the ability to define a robust responder index based on improvement in disease activity without worsening of the overall condition or the development of significant disease activity in new organ systems.
doi:10.1002/art.24698
PMCID: PMC2748175  PMID: 19714615
18.  Type I interferon correlates with serological and clinical manifestations of SLE 
Annals of the Rheumatic Diseases  2005;64(12):1692-1697.
Background: Systemic lupus erythematosus (SLE) is an autoimmune disease affecting multiple organ systems triggered by the production of autoantibodies. Previous clinical studies in humans and murine models suggest that type I interferons (IFNs) are important for the initiation and potentiation of SLE activity.
Methods: 65 consecutive patients with SLE were identified from the University of California, San Francisco Lupus Clinic with moderate-severe disease activity. 94 serological samples were collected. Type I IFN levels and the ability of plasma to induce expression of several surface markers of dendritic cell maturation were measured.
Results: Type I IFN levels correlated with the presence of cutaneous manifestations, and there was a trend towards correlation with renal disease. No correlation was found between type I IFN levels and neurological disease. Type I IFN levels correlated positively with the SLEDAI score and anti-dsDNA levels and inversely with C3 levels. Interestingly, type I IFN levels were highest in African American patients. SLE plasma also induced the expression of MHC class I, CD38, and CD123 on monocytes, and was blocked by the addition of a monoclonal antibody to IFNAR1.
Conclusions: The pathogenic role of type I IFN is suggested by the induction of cell surface markers for dendritic cell maturation. The potential therapeutic utility of antibodies directed to either type I IFN or IFNAR1/IFNAR2 may be of interest in further studies.
doi:10.1136/ard.2004.033753
PMCID: PMC1755300  PMID: 15843451
19.  Protection of Irf5-deficient mice from pristane-induced lupus involves altered cytokine production and class switching 
European journal of immunology  2012;42(6):1477-1487.
Summary
Polymorphisms in the transcription factor interferon (IFN) regulatory factor 5 (IRF5) have been identified that show strong association with increased risk of developing the autoimmune disease systemic lupus erythematosus (SLE). A potential pathologic role for IRF5 in SLE development is supported by the fact that increased IRF5 mRNA and protein abundance are observed in primary blood cells of SLE patients that correlate with increased risk of developing the disease. Here, we demonstrate that IRF5 is required for pristane-induced SLE via its ability to control multiple facets of autoimmunity. We show that IRF5 has a distinct influence on pathological hypergammaglobulinemia and provide evidence for its role in regulating IgG1 class switching and antigen specificity. Examination of in vivo cytokine expression (and autoantibody production) identified an imbalance in Irf5−/− mice favoring Th2 polarization. In addition, we provide clear evidence that loss of Irf5 significantly weakens the in vivo type I IFN signature critical for disease pathogenesis in this model of murine lupus. Together, these findings demonstrate the global effect that IRF5 has on autoimmunity and provides significant new insight into how overexpression of IRF5 in blood cells of SLE patients may contribute to disease pathogenesis.
doi:10.1002/eji.201141642
PMCID: PMC3684952  PMID: 22678902
interferon regulatory factor 5 (IRF5, IRF-5); systemic lupus erythematosus (SLE); autoantibody; type I interferon; Th2
20.  Serum Type I Interferon Activity Is Dependent on Maternal Diagnosis in Anti-SSA/Ro–Positive Mothers of Children With Neonatal Lupus 
Arthritis and rheumatism  2008;58(2):541-546.
Objective
The type I interferon (IFN) pathway is activated in many patients with systemic lupus erythematosus (SLE), and high serum levels of IFN are associated with anti-SSA/Ro autoantibodies. To investigate the clinical features associated with type I IFN production in vivo, we compared serum IFN activity in individuals with anti-SSA/Ro antibodies who were asymptomatic with that in individuals with clinical manifestations of SLE or Sjögren's syndrome (SS).
Methods
Antibody-positive sera from 84 mothers of children with manifestations of neonatal lupus were studied for type I IFN activity, using a functional reporter cell assay. Maternal health status was characterized as asymptomatic, SS, SLE, pauci-SLE, or pauci-SS, based on a screening questionnaire, telephone interview, and review of medical records. The prefix “pauci-” indicates symptoms insufficient for a formal classification of the disease.
Results
Only 4% of asymptomatic mothers had high serum type I IFN activity, compared with 73% with pauci-SLE (P = 5.7 × 10−5), 35% with SLE (P = 0.011), and 32% of patients with SS (P = 0.032). One of the 4 patients with pauci-SS had high levels of IFN. The majority of patients for whom longitudinal data were available had stable type I IFN activity over time, and changes in IFN activity were not clearly accompanied by changes in the clinical diagnosis.
Conclusion
Patients with SLE, patients with pauci-SLE, and patients with SS are more likely to have high serum IFN activity than asymptomatic individuals with SSA/Ro autoantibodies, suggesting that these autoantibodies are insufficient for activation of the type I IFN pathway, and that disease-specific factors are important for type I IFN generation in vivo.
doi:10.1002/art.23191
PMCID: PMC2755051  PMID: 18240214
21.  Plasmacytoid dendritic cells and C1q differentially regulate inflammatory gene induction by lupus immune complexes1 
Immune complexes (ICs) play a pivotal role in causing inflammation in systemic lupus erythematosus (SLE)3. Yet, it remains unclear what the dominant blood cell type(s) and inflammation related gene programs stimulated by lupus ICs are. To address these questions, we exposed normal human peripheral blood mononuclear cells (PBMCs) or CD14+ isolated monocytes to SLE ICs in the presence or absence of C1q and performed microarray analysis and other tests for cell activation. By microarray analysis, we identified genes and pathways regulated by SLE ICs that are both type I IFN dependent and independent. We also found that C1q containing ICs markedly reduced expression of the majority of IFN-response genes and also influenced the expression of multiple other genes induced by SLE ICs. Surprisingly, IC activation of isolated CD14+ monocytes did not upregulate CD40 and CD86 and only modestly stimulated inflammatory gene expression. However, when monocyte subsets were purified and analyzed separately, the low abundance CD14dim (‘patrolling’) subpopulation was more responsive to ICs. These observations demonstrate the importance of plasmacytoid dendritic cells (pDCs), CD14dim monocytes and C1q as key regulators of inflammatory properties of ICs and identify many pathways through which they act.
doi:10.4049/jimmunol.1102797
PMCID: PMC3238790  PMID: 22147767
Immune complexes; interferon alpha; plasmacytoid dendritic cells; monocytes; systemic lupus erythematosus; gene regulation; complement
22.  Type I interferon signature is high in lupus and neuromyelitis optica but low in multiple sclerosis 
Objective
Neuromyelitis optica (NMO) is characterized by selective inflammation of the spinal cord and optic nerves but is distinct from multiple sclerosis (MS). Interferon (IFN)-β mitigates disease activity in MS, but is controversial in NMO, with a few reports of disease worsening after IFN-β therapy in this highly active disease. In systemic lupus erythematosus (SLE), IFNs adversely affect disease activity. This study examines for the first time whether serum IFN-α/β activity and IFN-β-induced responses in peripheral blood mononuclear cells (MNC) are abnormally elevated in NMO, as they are in SLE, but contrast to low levels in MS.
Methods
Serum type I IFN-α/β activity was measured by a previously validated bioassay of 3 IFN-stimulated genes (RT-PCR sensitivity, 0.1 U/ml) rather than ELISA, which has lower sensitivity and specificity for measuring serum IFNs. IFN responses in PBMNC were assessed by in vitro IFN-β-induced activation of phospho-tyrosine-STAT1 and phospho-serine-STAT1 transcription factors, and MxA proteins using Western blots.
Results
Serum IFN-α/β activity was highest in SLE patients, followed by healthy subjects and NMO, but was surprisingly low in therapy-naïve MS. In functional assays in vitro, IFN-β-induced high levels of P-S-STAT1 in NMO and SLE, but not in MS and controls. IFN-β-induced MxA protein levels were elevated in NMO and SLE compared to MS.
Conclusions
Serum IFN activity and IFN-β-induced responses in PBMNC are elevated in SLE and NMO patients versus MS. This argues for similarities in pathophysiology between NMO and SLE and provides an explanation for IFN-induced disease worsening in NMO.
doi:10.1016/j.jns.2011.09.032
PMCID: PMC3910514  PMID: 22036215
NMO; MS; SLE; Interferon; STAT1; MxA
23.  SLE Peripheral Blood B Cell, T Cell and Myeloid Cell Transcriptomes Display Unique Profiles and Each Subset Contributes to the Interferon Signature 
PLoS ONE  2013;8(6):e67003.
Systemic lupus erythematosus (SLE) is a chronic autoimmune disease that is characterized by defective immune tolerance combined with immune cell hyperactivity resulting in the production of pathogenic autoantibodies. Previous gene expression studies employing whole blood or peripheral blood mononuclear cells (PBMC) have demonstrated that a majority of patients with active disease have increased expression of type I interferon (IFN) inducible transcripts known as the IFN signature. The goal of the current study was to assess the gene expression profiles of isolated leukocyte subsets obtained from SLE patients. Subsets including CD19+ B lymphocytes, CD3+CD4+ T lymphocytes and CD33+ myeloid cells were simultaneously sorted from PBMC. The SLE transcriptomes were assessed for differentially expressed genes as compared to healthy controls. SLE CD33+ myeloid cells exhibited the greatest number of differentially expressed genes at 208 transcripts, SLE B cells expressed 174 transcripts and SLE CD3+CD4+ T cells expressed 92 transcripts. Only 4.4% (21) of the 474 total transcripts, many associated with the IFN signature, were shared by all three subsets. Transcriptional profiles translated into increased protein expression for CD38, CD63, CD107a and CD169. Moreover, these studies demonstrated that both SLE lymphoid and myeloid subsets expressed elevated transcripts for cytosolic RNA and DNA sensors and downstream effectors mediating IFN and cytokine production. Prolonged upregulation of nucleic acid sensing pathways could modulate immune effector functions and initiate or contribute to the systemic inflammation observed in SLE.
doi:10.1371/journal.pone.0067003
PMCID: PMC3691135  PMID: 23826184
24.  Monocyte surface expression of Fcγ receptor RI (CD64), a biomarker reflecting type-I interferon levels in systemic lupus erythematosus 
Introduction
More than half of systemic lupus erythematosus (SLE) patients show evidence of excess type I interferon (IFN-I) production, a phenotype associated with renal disease and certain autoantibodies. However, detection of IFN-I proteins in serum is unreliable, and the measurement of interferon-stimulated gene (ISG) expression is expensive and time consuming. The aim of this study was to identify a surrogate marker for IFN-I activity in clinical samples for monitoring disease activity and response to therapy.
Methods
Monocyte surface expression of Fcγ receptors (FcγRs), chemokine receptors, and activation markers were analyzed with flow cytometry in whole blood from patients with SLE and healthy controls. FcγR expression also was measured in peripheral blood mononuclear cells (PBMCs) from healthy controls cultured with Toll-like receptor (TLR) agonists, cytokines, or serum from SLE patients. Expression of ISGs was analyzed with real-time PCR.
Results
Circulating CD14+ monocytes from SLE patients showed increased surface expression of FcγRI (CD64). The mean fluorescent intensity of CD64 staining correlated highly with the ISG expression (MX1, IFI44, and Ly6E). In vitro, IFN-I as well as TLR7 and TLR9 agonists, induced CD64 expression on monocytes from healthy controls. Exposure of monocytes from healthy controls to SLE sera also upregulated the expression of CD64 in an IFN-I-dependent manner. Decreased CD64 expression was observed concomitant with the reduction of ISG expression after high-dose corticosteroid therapy.
Conclusions
Expression of CD64 on circulating monocytes is IFN-I inducible and highly correlated with ISG expression. Flow-cytometry analysis of CD64 expression on circulating monocytes is a convenient and rapid approach for estimating IFN-I levels in SLE patients.
doi:10.1186/ar3017
PMCID: PMC2911874  PMID: 20478071
25.  Role of interleukin 10 in the B lymphocyte hyperactivity and autoantibody production of human systemic lupus erythematosus 
Interleukin-10 (IL-10) is produced at a high level by B lymphocytes and monocytes of patients with systemic lupus erythematosus (SLE). In the present work, we analyzed whether this increased production of IL-10 contributed to the abnormal production of immunoglobulins (Ig) and of autoantibodies in SLE. The role of IL-10 was compared with that of IL- 6, another cytokine suspected to play a role in these abnormalities. The spontaneous in vitro production of IgM, IgG, and IgA by peripheral blood mononuclear cells from SLE patients was weakly increased by recombinant IL (rIL)-6, but strongly by rIL-10. This production was not significantly affected by an anti-IL-6 mAb but was decreased by an anti- IL-10 mAb. We then tested the in vivo effect of these antibodies in severe combined immunodeficiency mice injected with PBMC from SLE patients. The anti-IL-6 mAb did not significantly affect the serum concentration of total human IgG and of anti-double-stranded DNA IgG in the mice. In contrast, the anti-IL-10 mAb strongly inhibited the production of autoantibodies, and, to a lesser extent, that of total human IgG. These results indicate that the Ig production by SLE B lymphocytes is largely IL-10 dependent, and that the increased production of IL-10 by SLE B lymphocytes and monocytes may represent a critical mechanism in the emergence of the autoimmune manifestations of the disease.
PMCID: PMC2191898  PMID: 7869046

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